Secretion from activated platelets is thought to be a key step in hemostasis as well as in the post-thrombus sequellae of wound healing. Controlling platelet exocytosis offers a unique therapeutic opportunity to manipulate the microenvironment formed at the site of vascular damage. However, our understanding of the machinery that facilitates platelet granule release is still not sufficient to identify valuable drug targets. Our proposed studies seek to rectify this by delineating the molecular events required for platelet secretion. From past reports, it is clear that specific Soluble NSF Attachment Protein Receptors (v- and t-SNAREs) are required for platelet secretion. With this established, our goals are now expanded to define the roles of secretion in hemostasis and to identify the mechanisms by which the exocytosis machinery is regulated. Two Specific Aims are proposed: 1) To assess the role of the v-SNARE, VAMP-8, in platelet function. Our work demonstrated that VAMP-8/endobrevin is required for platelet exocytosis. Aim 1 will complete the characterization of this v-SNARE's role using specific experiments to define the in vivo and in vitro phenotypes of VAMP-8-/- platelets. Other experiments will address the sub-platelet distribution of VAMP-8 and characterize its interacting partners. 2) To identify and characterize the SNARE regulators that interact with and control the syntaxin t-SNAREs in platelets. Past studies assigned roles for SNAP-23, syntaxin 2, and 4. This aim focuses on defining roles for known t-SNAREs regulators and identifying novel ones. The syntaxin-regulators Munc18, Munc13, DOC2, and tomosyn will be studied using biochemical techniques and a permeabilized platelet secretion assay. The experiments will expand our understanding of how these proteins mediate SNARE pairing and subsequent membrane fusion. In summary, the information gained from this proposal will identify new therapeutic targets to control hyperactive platelets and will provide diagnostic reagents to determine why hypoactive platelets are defective. PUBLIC HEALTH RELEVANCE: Strokes and heart attacks are major killers in the United States. In both cases, the damage is caused by a loss of blood flow, usually because a blood clot has formed in the wrong place. Understanding all aspects of clot formation is critical to treating this problem. This application focuses on one aspect of clot formation, platelets. Specifically this application focuses on how platelets secrete components needed for clot formation. This work is of critical important because it will direct the development of future therapeutic agents needed to decrease the risk of spurious clot formation. [unreadable] [unreadable] [unreadable] [unreadable]